NADPH-d and Fos reactivity in the rat spinal cord following experimental spinal cord injury and embryonic neural stem cell transplantation

AimsThe aim of this study is to determine the role of nitric oxide (NO) in neuropathic pain and the effect of embryonic neural stem cell (ENSC) transplantation on NO content in rat spinal cord neurons following spinal cord injury (SCI).Main methodsNinety adult male Sprague–Dawley rats were divided into 3 groups (n = 30, each): control (laminectomy), SCI (hemisection at T12–T13 segments) and SCI + ENSC. Each group was further divided into sub-groups (n = 5 each) based on the treatment substance (L-NAME, 75 mg/kg/i.p.; l-arginine, 225 mg/kg/i.p.; physiological saline, SF) and duration (2 h for acute and 28 days for chronic groups). Pain was assessed by tail flick and Randall–Selitto tests. Fos immunohistochemistry and NADPH-d histochemistry were performed in segments 2 cm rostral and caudal to SCI.Key findingsTail-flick latency time increased in both acute and chronic L-NAME groups and increased in acute and decreased in chronic l-arginine groups. The number of Fos (+) neurons decreased in acute and chronic L-NAME and decreased in acute l-arginine groups. Following ENSC, Fos (+) neurons did not change in acute L-NAME but decreased in the chronic L-NAME groups, and decreased in both acute and chronic l-arginine groups. NADPH-d (+) neurons decreased in acute L-NAME and increased in l-arginine groups with and without ENSC transplantation.SignificanceThis study confirms the role of NO in neuropathic pain and shows an improvement following ENSC transplantation in the acute phase, observed as a decrease in Fos(+) and NADPH-d (+) neurons in spinal cord segments rostral and caudal to injury.     

Acute and chronic tactile sensory testing after spinal cord injury in rats

Spinal cord injury (SCI) impairs sensory systems causing allodynia. To identify cellular and molecular causes of allodynia, sensitive and valid sensory testing in rat SCI models is needed. However, until recently, no single testing approach had been validated for SCI so that standardized methods have not been implemented across labs. Additionally, available testing methods could not be implemented acutely or when severe motor impairments existed, preventing studies of the development of SCI-induced allodynia(3). Here we present two validated sensory testing methods using von Frey Hair (VFH) monofilaments which quantify changes in tactile sensory thresholds after SCI. One test is the well-established Up-Down test which demonstrates high sensitivity and specificity across different SCI severities when tested chronically. The other test is a newly-developed dorsal VFH test that can be applied acutely after SCI when allodynia develops, prior to motor recovery. Each VFH monofilament applies a calibrated force when touched to the skin of the hind paw until it bends. In the up-down method, alternating VFHs of higher or lower forces are used on the plantar L5 dermatome to delineate flexor withdrawal thresholds. Successively higher forces are applied until withdrawal occurs then lower force VFHs are used until withdrawal ceases. The tactile threshold reflects the force required to elicit withdrawal in 50% of the stimuli. For the new test, each VFH is applied to the dorsal L5 dermatome of the paw while the rat is supported by the examiner. The VFH stimulation occurs in ascending order of force until at least 2 of 3 applications at a given force produces paw withdrawal. Tactile sensory threshold is the lowest force to elicit withdrawal 66% of the time. Acclimation, testing and scoring procedures are described. Aberrant trials that require a retest and typical trials are defined. Animal use was approved by Ohio State University Animal Care and Use Committee.     

Target populations for first-in-human embryonic stem cell research in spinal cord injury

Geron recently announced that it had begun enrolling patients in the world's first-in-human clinical trial involving cells derived from human embryonic stem cells (hESCs). This trial raises important questions regarding the future of hESC-based therapies, especially in spinal cord injury (SCI) patients. We address some safety and efficacy concerns with this research, as well as the ethics of fair subject selection. We consider other populations that might be better for this research: chronic complete SCI patients for a safety trial, subacute incomplete SCI patients for an efficacy trial, and perhaps primary progressive multiple sclerosis (MS) patients for a combined safety and efficacy trial.     

Icariin-treated human umbilical cord mesenchymal stem cells decrease chronic liver injury in mice

Human umbilical cord mesenchymal stem cells (hUMSCs) have been shown to have multiple differentiation potentials. However, a key problem is that only a small number of hUMSCs can migrate to damaged tissue after transplantation. According to “The Theory of Kidney Essence” in Traditional Chinese Medicine, some traditional Chinese medicines used for tonifying the kidneys can be applied in promoting the differentiation and migration of stem cells in vivo. Our previous study demonstrated that icariin (ICA) could up-regulate the pluripotent genes of hUMSCs in vitro and induce cell migration in mice in an acute kidney injury model in vivo. The aim of this study was to investigate the effects of ICA-induced hUMSCs in chronic liver injury (CLI) caused by carbon tetrachloride (CCl₄). CLI was induced by intraperitoneal injection of CCl₄. ICA-treated hUMSCs were transplanted via intra-venous injection. The animals were followed for survival, biochemistry analysis and pathology. The results show that ICA-treated hUMSCs accelerate the recovery of liver function in mice with CLI. In addition, ICA-treated hUMSCs increase the anti-oxidant activities in liver and prevent the progression to hepatic fibrosis. Moreover, ICA induces the migration of hUMSCs to the injured liver tissue. In conclusion, these data demonstrate that ICA-treated hUMSCs exhibit recovery and protective properties in the mice model of CCl₄-induced CLI.     

Human umbilical cord blood-derived stromal cells prevent graft-versus-host disease in mice following haplo-identical stem cell transplantation

Background aimsHuman umbilical cord blood-derived stromal cells (hUCBDSC) comprise a novel population of CD34⁺ cells that has been isolated in our laboratory. They have been shown previously not only to be non-immunogenic but also to exert immunosuppressive effects on xenogenic T cells in vitro. This study investigated the role of hUCBDSC in immunomodulation in an acute graft-versus-host disease (GvHD) mouse model after haplo-identical stem cell transplantationMethodsAcute GvHD was induced in recipient (B6 × BALB/c)F₁ mice by irradiation (750 cGy) followed by infusion of bone marrow cells and splenocytes from donor C57BL/6 mice. hUCBDSC were co-transplanted in the experimental group. The survival time, body weight and clinical and histopathologic scores were recorded after transplantation. The expression of surface markers [major histocompatibility complex (MHC) I, MHC II, CD80 and CD86] on CD11c⁺ dendritic cells (DC), and the percentage of CD4⁺ regulatory T cells (Treg), in the spleens of recipient mice were examined by flow cytometryResultsThe survival time was significantly prolonged, and the clinical and histopathologic scores were reduced in mice co-transplanted with hUCBDSC. The expression levels of the surface markers on DC were significantly lower in mice transplanted with hUCBDSC compared with those without. The proportion of CD4⁺ Treg in the spleen was also increased in mice transplanted with hUCBDSCConclusionsThese results from a GvHD mouse model are in agreement with previous in vitro findings, suggesting that hUCBDSC possess immunosuppressive properties and may act via influencing DC and CD4⁺ Treg.     

3D spheroids of human placenta-derived mesenchymal stem cells attenuate spinal cord injury in mice

Mesenchymal stem cell (MSC) is an absorbing candidate for cell therapy in treating spinal cord injury (SCI) due to its great potential for multiple cell differentiation, mighty paracrine secretion as well as vigorous immunomodulatory effect, of which are beneficial to the improvement of functional recovery post SCI. However, the therapeutic effects of MSC on SCI have been limited because of the gradual loss of MSC stemness in the process of expanding culture. Therefore, in this study, we aimed to maintain those beneficial properties of MSC via three-dimensional spheroid cell culture and then compared them with conventionally-cultured MSCs in the treatment of SCI both in vitro and in vivo with the aid of two-photon microscope. We found that 3D human placenta-derived MSCs (3D-HPMSCs) demonstrated a significant increase in secretion of anti-inflammatory factors and trophic factors like VEGF, PDGF, FGF via QPCR and Bio-Plex assays, and showed great potentials on angiogenesis and neurite morphogenesis when co-cultured with HUVECs or DRGs in vitro. After transplantation into the injured spinal cord, 3D-HPMSCs managed to survive for the entire experiment and retained their advantageous properties in secretion, and exhibited remarkable effects on neuroprotection by minimizing the lesion cavity, inhibiting the inflammation and astrogliosis, and promoting angiogenesis. Further investigation of axonal dieback via two-photon microscope indicated that 3D-HPMSCs could effectively alleviate axonal dieback post injury. Further, mice only treated with 3D-HPMSCs obtained substantial improvement of functional recovery on electrophysiology, BMS score, and Catwalk analysis. RNA sequencing suggested that the 3D-HPMSCs structure organization-related gene was significantly changed, which was likely to potentiate the angiogenesis and inflammation regulation after SCI. These results suggest that 3D-HPMSCs may hold great potential for the treatment of SCI.Subject terms: Spinal cord injury, Mesenchymal stem cells     

Acrolein involvement in sensory and behavioral hypersensitivity following spinal cord injury in the rat

Growing evidence suggests that oxidative stress, as associated with spinal cord injury (SCI), may play a critical role in both neuroinflammation and neuropathic pain conditions. The production of the endogenous aldehyde acrolein, following lipid peroxidation during the inflammatory response, may contribute to peripheral sensitization and hyperreflexia following SCI via the TRPA1‐dependent mechanism. Here, we report that there are enhanced levels of acrolein and increased neuronal sensitivity to the aldehyde for at least 14 days after SCI. Concurrent with injury‐induced increases in acrolein concentration is an increased expression of TRPA1 in the lumbar (L3–L6) sensory ganglia. As proof of the potential pronociceptive role for acrolein, intrathecal injections of acrolein revealed enhanced sensitivity to both tactile and thermal stimuli for up to 10 days, supporting the compound's pro‐nociceptive functionality. Treatment of SCI animals with the acrolein scavenger hydralazine produced moderate improvement in tactile responses as well as robust changes in thermal sensitivity for up to 49 days. Taken together, these data suggest that acrolein directly modulates SCI‐associated pain behavior, making it a novel therapeutic target for preclinical and clinical SCI as an analgesic.

     

Transplantation of porcine embryonic stem cells and their derived neuronal progenitors in a spinal cord injury rat model

Background aimsThe purpose of this study was to investigate therapeutic potential of green fluorescent protein expressing porcine embryonic stem (pES/GFP⁺) cells in A rat model of spinal cord injury (SCI).MethodsUndifferentiated pES/GFP⁺ cells and their neuronal differentiation derivatives were transplanted into the contused spinal cord of the Long Evans rat, and in situ development of the cells was determined by using a live animal fluorescence optical imaging system every 15 days. After pES/GFP⁺ cell transplantation, the behavior functional recovery of the SCI rats was assessed with the Basso, Beattie, and Bresnahan Locomotor Rating Scale (BBB scale), and the growth and differentiation of the grafted pES/GFP⁺ cells in the SCI rats were analyzed by immunohistochemical staining.ResultsThe relative green fluorescent protein expression level was decreased for 3 months after transplantation. The pES/GFP⁺-derived cells positively stained with neural specific antibodies of anti-NFL, anti-MBP, anti-SYP and anti-Tuj 1 were detected at the transplanted position. The SCI rats grafted with the D18 neuronal progenitors showed a significant functional recovery of hindlimbs and exhibited the highest BBB scale score of 15.20 ± 1.43 at week 24. The SCI rats treated with pES/GFP⁺-derived neural progenitors demonstrated a better functional recovery.ConclusionsTransplantation of porcine embryonic stem (pES)-derived D18 neuronal progenitors has treatment potential for SCI, and functional behavior improvement of grafted pES-derived cells in SCI model rats suggests the potential for further application of pES cells in the study of replacement medicine and functionally degenerative pathologies.     

Engraftment,migration and differentiation of neural stem cells in the rat spinal cord following contusion injury

Background aimsSpinal cord injury is a devastating injury that impacts drastically on the victim's quality of life. Stem cells have been proposed as a therapeutic strategy. Neural stem (NS) cells have been harvested from embryonic mouse forebrain and cultured as adherent cells. These NS cells express markers of neurogenic radial glia.MethodsMouse NS cells expressing green fluorescent protein (GFP) were transplanted into immunosupressed rat spinal cords following moderate contusion injury at T9. Animals were left for 2 and 6 weeks then spinal cords were fixed, cryosectioned and analyzed. Stereologic methods were used to estimate the volume and cellular environment of the lesions. Engraftment, migration and differentiation of NS cells were also examined.ResultsNS cells integrated well into host tissue and appeared to migrate toward the lesion site. They expressed markers of neurons, astrocytes and oligodendrocytes at 2 weeks post-transplantation and markers of neurons and astrocytes at the 6-week time-point. NS cells appeared to have a similar morphologic phenotype to radial glia, in particular at the pial surface.ConclusionsAlthough no functional recovery was observed using the Basso Beattie Bresnahan (BBB) locomotor rating scale, NS cells are a potential cellular therapy for treatment of injured spinal cord. They may be used as delivery vehicles for therapeutic proteins because they show an ability to migrate toward the site of a lesion. They may also be used to replace lost or damaged neurons and oligodendrocytes.     

胚胎神经干细胞移植及胶质细胞源性神经营养因子对大鼠脊髓损伤的修复作用

将胚胎神经干细胞(neural stem cells,NSCs)移植至成年大鼠损伤的脊髓,观察移植后NSCs的存活、迁移以及损伤后的功能恢复。实验结果显示：动物NSCs移植4周后,斜板实验平均角度和运动评分结果比对照组均有明显增高(P＜0．05),而脊髓损伤(spinal cord injury,SCI)处的空洞面积显著减小(P＜0．05);在NSCs中加入胶质细胞源性的神经营养因子(glial cell line-derived neurotrophic factor,GDNF)后,上述改变更加显著。移植后的NSCs不仅能存活,而且向损伤的头端和尾端迁移达3mm之远。这些结果表明,移植的NSCs不仅可以存活、迁移,还可减小SCI空洞面积,促进动物神经功能的恢复;此外,我们的结果还表明GDNF对SCI功能恢复有促进作用。     

Multipotent hair follicle stem cells promote repair of spinal cord injury and recovery of walking function

The mouse hair follicle is an easily accessible source of actively growing, pluripotent adult stem cells. C57BL transgenic mice, labeled with the fluorescent protein GFP, afforded follicle stem cells whose fate could be followed when transferred to recipient animals. These cells appear to be relatively undifferentiated since they are positive for the stem cell markers nestin and CD34 but negative for the keratinocyte marker keratin 15. These hair follicle stem cells can differentiate into neurons, glia, keratinocytes, smooth muscle cells, and melanocytes in vitro. Implanting hair follicle stem cells into the gap region of severed sciatic or tibial nerves greatly enhanced the rate of nerve regeneration and restoration of nerve function. The transplanted follicle cells transdifferentiated mostly into Schwann cells, which are known to support neuron regrowth. The treated mice regained the ability to walk essentially normally. In the present study, we severed the thoracic spinal chord of C57BL/6 immunocompetent mice and transplanted GFP-expressing hair follicle stem cells to the injury site. Most of the transplanted cells also differentiated into Schwann cells that apparently facilitated repair of the severed spinal cord. The rejoined spinal cord reestablished extensive hind-limb locomotor performance. These results suggest that hair follicle stem cells can promote the recovery of spinal cord injury. Thus, hair follicle stem cells provide an effective accessible, autologous source of stem cells for the promising treatment of peripheral nerve and spinal cord injury.     

Transplantation of apoptosis-resistant embryonic stem cells into the injured rat spinal cord

Murine embryonic stem cells were induced to differentiate into neural lineage cells by exposure to retinoic acid. Approximately one million cells were transplanted into the lesion site in the spinal cords of adult rats which had received moderate contusion injuries 9 days previously. One group received transplants of cells genetically modified to over-express bcl-2, which codes for an anti-apoptotic protein. A second group received transplants of the wild-type ES cells from which the bcl-2 line was developed. In the untransplanted control group, only medium was injected. Locomotor abilities were assessed using the Basso, Beattie and Bresnahan (BBB) rating scale for 6 weeks. There was no incremental locomotor improvement in either transplant group when compared to control over the survival period. Morbidity and mortality were significantly more prevalent in the transplant groups than in controls. At the conclusion of the 6-week survival period, the spinal cords were examined. Two of six cords from the bcl-2 group and one of 12 cords from the wild-type group showed gross evidence of abnormal growths at the site of transplantation. No similar growth was seen in the control. Pathological examination of the abnormal cords showed very large numbers of undifferentiated cells proliferating at the injection site and extending up to 1.5?cm rostrally and caudally. These results suggest that transplanting KD3 ES cells, or apoptosis-resistant cells derived from the KD3 line, into the injured spinal cord does not improve locomotor recovery and can lead to tumor-like growth of cells, accompanied by increased debilitation, morbidity and mortality.     

Transplantation of apoptosis-resistant embryonic stem cells into the injured rat spinal cord

Murine embryonic stem cells were induced to differentiate into neural lineage cells by exposure to retinoic acid. Approximately one million cells were transplanted into the lesion site in the spinal cords of adult rats which had received moderate contusion injuries 9 days previously. One group received transplants of cells genetically modified to over-express bcl-2, which codes for an anti-apoptotic protein. A second group received transplants of the wild-type ES cells from which the bcl-2 line was developed. In the untransplanted control group, only medium was injected. Locomotor abilities were assessed using the Basso, Beattie and Bresnahan (BBB) rating scale for 6 weeks. There was no incremental locomotor improvement in either transplant group when compared to control over the survival period. Morbidity and mortality were significantly more prevalent in the transplant groups than in controls. At the conclusion of the 6-week survival period, the spinal cords were examined. Two of six cords from the bcl-2 group and one of 12 cords from the wild-type group showed gross evidence of abnormal growths at the site of transplantation. No similar growth was seen in the control. Pathological examination of the abnormal cords showed very large numbers of undifferentiated cells proliferating at the injection site and extending up to 1.5 cm rostrally and caudally. These results suggest that transplanting KD3 ES cells, or apoptosis-resistant cells derived from the KD3 line, into the injured spinal cord does not improve locomotor recovery and can lead to tumor-like growth of cells, accompanied by increased debilitation, morbidity and mortality.     

Sensory function above lesion level in spinal cord injury patients with and without pain

Patients with spinal cord injury (SCI) may or may not develop central neuropathic pain despite having cord lesions of apparently the same site, extension and nature. The consequences of the cord lesion in the central nervous system and the mechanisms underlying pain are unclear. In this study, we examined sensory detection and pain thresholds above injury level in 17 SCI patients with central neuropathic pain, in 18 SCI patients without neuropathic pain, and in 20 control subjects without injury and pain. The SCI pain group had significantly higher cold and warm detection thresholds compared with the SCI pain free group and controls and higher tactile detection thresholds compared with the SCI pain free group. No difference in pain or pain tolerance thresholds was seen among pain and pain free SCI patients. These data suggest changes in somatosensory function in dermatomes rostral to the segmental injury level linked to the presence of central neuropathic pain in SCI patients. The results are discussed in relation to current concepts of pain inhibitory and facilitating systems.     

Transplantation of neural stem cells into the spinal cord after injury

Thanks to advances in the stem cell biology of the central nervous system (CNS), the previously inconceivable regeneration of the damaged CNS is approaching reality. The availability of signals to induce the appropriate differentiation of the transplanted and/or endogenous neural stem cells (NSCs) as well as the timing of the transplantation are important for successful functional recovery of the damaged CNS. Because the immediately post-traumatic microenvironment of the spinal cord is in an acute inflammatory stage, it is not favorable for the survival and differentiation of NSC transplants. On the other hand, in the chronic stage after injury, glial scars form in the injured site that inhibit the regeneration of neuronal axons. Thus, we believe that the optimal timing of transplantation is 1-2 weeks after injury.     

Sensory function above lesion level in spinal cord injury patients with and without pain

Patients with spinal cord injury (SCI) may or may not develop central neuropathic pain despite having cord lesions of apparently the same site, extension and nature. The consequences of the cord lesion in the central nervous system and the mechanisms underlying pain are unclear. In this study, we examined sensory detection and pain thresholds above injury level in 17 SCI patients with central neuropathic pain, in 18 SCI patients without neuropathic pain, and in 20 control subjects without injury and pain. The SCI pain group had significantly higher cold and warm detection thresholds compared with the SCI pain free group and controls and higher tactile detection thresholds compared with the SCI pain free group. No difference in pain or pain tolerance thresholds was seen among pain and pain free SCI patients. These data suggest changes in somatosensory function in dermatomes rostral to the segmental injury level linked to the presence of central neuropathic pain in SCI patients. The results are discussed in relation to current concepts of pain inhibitory and facilitating systems.     

Transplanted embryonic stem cells survive, differentiate and promote recovery in injured rat spinal cord

Transplantation approaches using cellular bridges, fetal central nervous system cells, fibroblasts expressing neurotrophin-3 (ref. 6), hybridoma cells expressing inhibitory protein-blocking antibodies, or olfactory nerves ensheathing glial cells transplanted into the acutely injured spinal cord have produced axonal regrowth or functional benefits. Transplants of rat or cat fetal spinal cord tissue into the chronically injured cord survive and integrate with the host cord, and may be associated with some functional improvements. In addition, rats transplanted with fetal spinal cord cells have shown improvements in some gait parameters, and the delayed transplantation of fetal raphe cells can enhance reflexes. We transplanted neural differentiated mouse embryonic stem cells into a rat spinal cord 9 days after traumatic injury. Histological analysis 2-5 weeks later showed that transplant-derived cells survived and differentiated into astrocytes, oligodendrocytes and neurons, and migrated as far as 8 mm away from the lesion edge. Furthermore, gait analysis demonstrated that transplanted rats showed hindlimb weight support and partial hindlimb coordination not found in 'sham-operated' controls or control rats transplanted with adult mouse neocortical cells.     

Glial and axonal regeneration following spinal cord injury

Spinal cord injury (SCI) has been regarded clinically as an irreversible damage caused by tissue contusion due to a blunt external force. Past research had focused on the analysis of the pathogenesis of secondary injury that extends from the injury epicenter to the periphery, as well as tissue damage and neural cell death associated with secondary injury. Recent studies, however, have proven that neural stem (progenitor) cells are also present in the brain and spinal cord of adult mammals including humans. Analyses using spinal cord injury models have also demonstrated active dynamics of cells expressing several stem cell markers, and methods aiming at functional reconstruction by promoting the potential self-regeneration capacity of the spinal cord are being explored. Furthermore, reconstruction of the neural circuit requires not only replenishment or regeneration of neural cells but also regeneration of axons. Analysis of the tissue microenvironment after spinal cord injury and research aiming to remove axonal regeneration inhibitors have also made progress. SCI is one of the simplest central nervous injuries, but its pathogenesis is associated with diverse factors, and further studies are required to elucidate these complex interactions in order to achieve spinal cord regeneration and functional reconstruction.Key words: glia, regeneration, spinal cord, injury, axon